The present invention relates to novel vaccine adjuvants. administered, for example, parenterally, induces an immune response, including the production of antibodies. Antibodies are substances contained in the blood and other fluids of the body, as well as in the tissues, and which bind to the antigen to make it innocuous. Antibodies constitute one of the natural defense mechanisms of the body. They are highly specific and they can kill, bind or make innocuous the antigen which has induced their formation.
The antigen, in contact with the immune system, thus activates a complex series of cellular interactions to eliminate the antigen and/or to reestablish the preceding equilibrium.
Two of the characteristic features of antigens are their immunogenicity, that is, their capacity to induce an immune response in vivo (including the formation of specific antibodies), and their antigenicity, that is, their capacity to be selectively recognized by the antibodies whose origins are the antigens.
It is known that it is possible to stimulate the immune response deliberately by administrating a specific antigen by means of a vaccine. This procedure allows the development in the organism of a state of immunologic memory which ensures a more rapid and more effective response of the organism during subsequent contact with the antigen.
However, some antigens have only a weak immunogenicity and they induce an insufficient immune response to procure an effective protection for the organism.
The immunogenicity of an antigen can be increased by administering it in a mixture with substances, called adjuvants, which increase the response against the antigen either by directly acting on the immunological system or by modifying the pharmacokinetic characteristics of the antigen and by thus increasing the interaction time between the latter with the immune system.
The most widespread adjuvants are, on the one hand, Freund""s adjuvant, an emulsion comprising dead mycobacteria in a saline solution within mineral oil and, on the other hand, Freund""s incomplete adjuvant, which does not contain mycobacteria.
These adjuvants are capable of either increasing the intensity of the immune response to the antigen or of producing an aspecific activation of the immune system.
However, the use of these adjuvants comprises drawbacks such as the formation of irritation or abscess at the point of injection. In addition, for these adjuvants to be effective, the concentration used must be greater than 50% of the injected volume, which limits the useful load of antigens which one can inject in one dose.
The high viscosity of these standard adjuvants based on oil and water make their use impractical because they are difficult to introduce into syringes and inject into the animals.
Another type of adjuvant which has been described comprises a solution of polyacrylic acid (Diamanstein et al., Z. Klin. Chem. Klin. Biochem., Vol. 8, pp. 632-636 (1970) and Diamanstein et al., Eur. J. Immunol., Vol. 1, pp. 335-339 (1971)). The advantage of this type of adjuvant is that it is less viscous than the conventional adjuvants based on mineral oil and water. It can therefore be manipulated and injected more easily. However, the efficacy of these adjuvants is not comparable to that of adjuvants based on water in mineral oil (W/O).
The purpose of the present invention is to propose an adjuvant for vaccines which is effective in a small concentration and without mineral oil.
This purpose is achieved by a vaccine adjuvant comprising an aqueous solution of polymers having anionic constitutive repeating units and hydrophobic constitutive repeating units.
The term anionic constitutive repeating units denotes, for the purposes of the present invention, monomer units, which constitute the polymer, containing groups capable of dissociating into water while forming anions.
Examples of such monomer units which are of use in the present invention to form the anionic constitutive repeating units are (selected from) acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylenesulfonic acid, vinylsulfuric acid, styrenesulfonic acid, vinylphenylsulfuric acid, 2-methacryloyloxyethanesulfonic acid, 3-methacryloyloxy-2-hydroxypropanesulfonic acid, 2-acryl-2-methylpropanesulfonic acid, 3-acrylamido-3-methylbutanoic acid, 3-methacrylamido-3-methylbutanoic acid, vinylphosphoric acid, 4-vinylbenzoic acid, 3-vinyloxypropane-1-sulfonic acid and N-vinylsuccimidic acid.
Preferably, the monomer units of this type are selected from acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylenesulfonic acid, vinylsulfuric acid and styrenesulfonic acid.
It is preferred for the monomer units of this type to be selected from acrylic acid, methacrylic acid, maleic acid and fumaric acid.
It is particularly preferred for the monomer units of this type to be acrylic acid units.
The term hydrophobic constitutive repeating units denotes, for the purposes of the present invention, monomer units, which constitute the polymer, containing exclusively hydrophobic groups, also called lipophilic, which do not dissociate in water.
Examples of such monomer units which are of use in the present invention to form the hydrophobic constitutive repeating units are (selected from) alkyl esters, cycloalkyl esters and hydroxyalkyl esters of the above-mentioned acids (acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylenesulfonic acid, vinylsulfuric acid, styrenesulfonic acid, vinylphenylsulfuric acid, 3-methacryloyloxy-2-hydroxypropanesulfonic acid, 2-methacryloyloxyethanesulfonic acid, 2-acryl-2-methylpropanesulfonic acid, 3-acrylamido-3-methylbutanoic acid, 3-methacrylamido-3-methylbutanoic acid, vinylphosphoric acid, 4-vinylbenzoic acid, 3-vinyloxypropane-1-sulfonic acid or N-vinylsuccimidic acid), and ethers (for example, methoxymethyl, ethoxyethyl, allyloxymethyl, 2-ethoxyethoxymethyl, benzyloxymethyl, cyclohexylmethyl, 1-ethoxyethyl, 2-ethoxyethyl, 2-butoxyethyl, methoxymethoxyethyl, methoxyethoxyethyl, 1-butoxypropyl, 1-ethoxybutyl, tetrahydrofurfuryl or furfuryl ethers).
Preferably, the monomer units of this type are selected from the alkyl esters of acrylic acid, methacrylic acid, maleic acid, fumaric acid, ethylenesulfonic acid, vinylsulfuric acid or styrenesulfonic acid.
Preferably, the monomer units of this type are selected from the alkyl esters of acrylic acid, methacrylic acid, maleic acid or fumaric acid whose alkyl group contains 4-8 carbon atoms.
It is particularly preferred for the monomer units of this type to be linear alkyl esters of acrylic acid whose alkyl group contains 4-8 carbon atoms.
It is particularly preferred for the adjuvants used according to the present invention to be aqueous solutions of polymers whose monomer units used to form the anionic constitutive repeating units consist of acrylic acid, and whose monomer units used to form the hydrophobic constitutive repeating units are selected from linear alkyl esters of acrylic acid whose alkyl group contains 4-8 carbon atoms.
The humoral response to the Vaccines which comprise an aqueous solution of polymers having anionic constitutive repeating units and hydrophobic constitutive repeating units is greater than the response induced by polymers having exclusively anionic constitutive repeating units, such as, for example, the polyacrylic acids.
Indeed, the efficacy of the adjuvants according to the present invention is comparable to that of the standard adjuvants based in water in mineral oil, whereas, in general, their toxicity is much lower.
The adjuvants according to the present invention therefore do not pose any instability problems , as do the standard adjuvants based on an emulsion of oil in water (O/W) or water in oil (W/O), because the latter are always sensitive to stabilizing factors such as the salt concentration, the temperature, etc., which is not the case for the adjuvants for the present invention. Their stability, in principle, corresponds to the stability of the polymers exclusively containing anionic constitutive repeating units, such as the polyacrylic acids.
One of the advantages of the adjuvant according to the present invention is that it is effective at a weak dose. It is therefore possible to increase the load of antigens per volume injected. In the vaccines based on W/O, the mineral oil occupies approximately 50% of the volume of the vaccine, whereas the volume fraction occupied by the adjuvants according to the present invention (for example, based on polyacrylic acids bound to hydrocarbon chains) can be decreased to approximately 10% of the volume of the vaccine.
According to a first advantageous embodiment, the molecular weight of the polymers is between 10 and 10,000 kD.
Advantageously, the molar ratio of hydrophobic constitutive repeating units and of the anionic constitutive repeating units is between 0.05 and 1.00 and, preferably between 0.10 and 0.40.
Preferably, the solubility of the polymers in water is at least 1 g/L.
According to another feature of the present invention, a process to obtain the polymer is described. The polymer can be obtained by one of the following processes:
1. copolymerization of anionic and hydrophobic monomers,
2. partial grafting of polymers,
3. partial hydrolysis of polymers, and
4. by an intermediate anhydride.
According to another preferred embodiment, a vaccine is proposed with a concentration of the polymer of 1-40 mg/mL of vaccine, preferably 4-24 mg/mL of vaccine, and more preferably 8-16 mg/mL of vaccine.
According to another aspect of the present invention, the vaccine comprises inactivated antigens of the Newcastle disease virus (NDV) and/or of the infectious bronchitis virus (IBV) for the vaccination of domestic animals.
The vaccines comprising an adjuvant based on polyacrylic acids bound to hydrocarbon chains are much more stable than the vaccines comprising an adjuvant based on a W/O or O/W emulsion, or based on water in mineral oil in water (W/O/W), because the adjuvant is a solution.
According to yet another feature of the present invention, the use of an aqueous solution of polymers is proposed, which has anionic constitutive repeating units and hydrophobic constitutive repeating units as adjuvant in vaccines.